BUSCADOR RECOLECTA

All images are super-resolution single confocal sections except B, which is a projection of super-resolution confocal sections. (A, B) In the trachea of wild-type embryos, Reb and Kkv do not colocalise, and they show a complementary pattern (A’-A”’) at the local subcellular level. (C) In salivary gland of embryos expressing Reb, the patterns of Kkv and Reb are complementary. (D) Models for the role of kkv and exp/reb in chitin deposition. Kkv oligomerises in complexes that localise to the apical membrane (as proposed in [2]). In the absence of exp/reb activity, Kkv can polymerise chitin from sugar monomers (discontinuous red lines), but it cannot translocate it because the channel is closed, and polymerised chitin remains in the cytoplasm. In addition, Kkv is not homogeneously distributed. Exp/Reb form a complex with other proteins, which localises to the apical membrane. The presence of Exp/Reb complex regulates Kkv apical distribution and activity. In model 1, we propose that a factor/s recruited by Exp/Reb (Factor X) can induce a posttranslation or conformational modification to Kkv protein that opens the channel promoting translocation of chitin fibers to the extracellular domain. In model 2, we propose that a factor/s recruited by Exp/Reb (Factor X’) can induce changes in membrane composition/curvature that will then promote a conformational change in Kkv that opens the channel to translocate chitin. These membrane changes lead to Kkv shedding extracellularly. In model 3, we propose that Exp/Reb complex can bind and relocalise Factor X”, which normally inhibits Kkv-translocating activity. This neutralises the activity of Factor X” allowing chitin translocation. Scale bars: 5 μm., Peer reviewed

All images are projections of confocal sections, of super-resolution microscopy. (A, B) Kkv localises apically in the trachea of wild-type embryos (A) and in absence of exp reb (B). (C, D) The localisation of Kkv is apical also in presence of exp ΔMH2 in trachea (C) and in presence of MH2-exp in salivary glands (D). (E, F) At stage 14, in wild-type embryos (E) and in embryos deficient for exp and reb (F), Kkv is present in the apical membrane and in many intracellular vesicles (yellow arrowheads). (G) At stage 16, in wild-type embryo, Kkv apical distribution follows the pattern of taenidial folds and intracellular vesicles are mostly absent. (H) At stage 16, in exp reb mutant embryos, Kkv is apical but shows altered distribution pattern. (I, J) At stage 15, in control embryos, Kkv pattern is apical and covers the whole membrane leaving minimal spatial gaps (I); instead, in exp reb mutant embryos, Kkv distribution changes to a less organised pattern at the apical membrane (J). (K) Three different types of spatial distribution within a selected area. The positions of the defined objects can be random and exhibit characteristics of attraction (clustered pattern) or repulsion (regular pattern). The F-Function tends to be larger (≈1) for clustered patterns and smaller (≈0) for regular. The G-Function tends to be smaller (≈0) for clustered and larger (≈1) for regular patterns. (L) Kkv punctae (magenta) on the apical cell area marked by Armadillo (green) in the trachea of a control embryo. (L’) Positions of Kkv punctae on the selected area marked by black dots. (L”) Random pattern of distribution for the same area created by the spatial statistics 2D/3D image analysis plugin. (M) The corresponding observed F and G functions (blue) are displayed above and below the reference simulated random distributions (black) and the 95% confidence interval (light gray), respectively, indicating a nonrandom spatial pattern. (N) SDI histogram for the F-Function of the control (blue) and the Df(exp reb) samples. A significant difference between the frequency distributions for each group of individuals has been observed. (Kolmogorov–Smirnov D = 0.5833, p < 0.05) (N’) SDI histogram for the G-Function of the control (blue) and the Df(exp reb) samples. Statistical analysis of the distributions did not reveal significant differences between the two groups of individuals for this parameter (Kolmogorov–Smirnov D = 0.25, p > 0.05). (O) Kkv punctae (magenta) on the apical cell area marked by Armadillo (green) in the trachea of a exp reb mutant embryo. (O’) Positions of Kkv punctae on the selected area marked by black dots. (O”) Random pattern of distribution for the same area created by the spatial statistics 2D/3D image analysis plugin. (P) The corresponding observed F and G functions (blue) are displayed above and below the reference simulated random distributions (black), respectively. Both curves largely overlap with the 95% confidence interval (light gray), indicating a tendency towards a random spatial pattern. (Q) Frequency distribution histograms for the nearest neighbour distances between Kkv punctae in control (blue) and exp reb mutant samples. The distribution of values between the two groups is found significantly different (Kolmogorov–Smirnov D = 0.2036, p < 0.005). The underlying data for quantifications can be found in the S1 Data. Scale bars A-J: 10 μm; L, O: 2 μm., Peer reviewed

All images are single confocal sections except A-C, which are projections of confocal sections. (A, A’) In trachea of wild-type embryos, Kkv is present in the apical region (blue arrows) and in intracellular vesicles (yellow arrowheads). (B) In kkv mutants unable to polymerise chitin, Kkv is not properly localised. (C) GFP-Kkv localises to the apical region (blue arrows) and in intracellular vesicles (yellow arrowheads). (D) When reb and GFP-kkv are coexpressed in salivary glands, Kkv is present in the apical membrane (blue arrow), in intracellular vesicles (yellow arrowhead), and also in punctae in the lumen (pink arrowheads). This is clearly observed in orthogonal sections (D’). (E) These luminal punctae corresponded to membranous structures. (F, F’) In contrast, when GFP-kkv is expressed alone, luminal punctae are absent, and Kkv is only found apically (blue arrow) and in intracellular vesicles (yellow arrowhead). (G) Luminal punctae (pink arrowheads) are also observed in the trachea of embryos overexpressing reb and GFP-kkv. (H) When endocytosis is prevented, the coexpression of reb and GFP-kkv in salivary glands still leads to formation of Kkv luminal punctae (pink arrowheads). (I) Quantifications of the number of intracellular Kkv vesicles in salivary glands when expressing GFP-kkv (I’), reb and GFP-kkv (I”), and GFP-kkvΔCC. n is the number of salivary glands analysed. The underlying data for quantifications can be found in the S1 Data. Scale bars: 10 μm., Peer reviewed

All images show salivary glands. (A, C, E-L, N, O) Show single confocal sections and (B, D, M) show projections of several sections. (A-A’) The concomitant expression of reb and GFP-kkv leads to luminal chitin deposition (blue arrow in orthogonal section in A’). (B, C) Coexpression of expΔMH2 and GFP-kkv produces intracellular chitin punctae, some of which partially colocalise with GFP-Kkv vesicles (yellow arrowhead) while others do not (red arrowhead). GFP-Kkv vesicles without chitin are also observed (green arrowhead). Note the accumulation of chitin in the apical domain (white arrow in C) that is not deposited extracellularly in the lumen (blue arrow in orthogonal section in C’). (D) In Rab5DN background, intracellular chitin punctae are still present (white arrowheads). (E-L) Analysis of the nature of GFP-Kkv vesicles and chitin punctae using markers Golgin245 (E-F), Hrs 27–4 (G-H), Arl8 (I-J), and Rab11 (K-L); arrowheads indicate colocalisation between Kkv and each specific marker. (M, N) All GFP-Kkv vesicles colocalise with the membrane marker CD4-mIFP (white arrowheads), and few of them also with chitin (orange arrowheads); single chitin punctae do not colocalise with CD4-mIFP (red arrowheads). (O-O”) Frames from live imaging movie show that partially colocalising GFP-Kkv and chitin punctae (yellow arrow) can separate from each other; however, many GFP-Kkv (green arrow) and chitin puncta (red arrow) do not colocalise. Scale bars A-D, M: 10 μm; E-L, N-N”‘: 1 μm; O-O”: 5 μm., Peer reviewed

(A) Schematic representation of Kkv protein (CD, catalytic domain; WGTRE; CC, coiled-coil domain). (B, C, G, I, J) Show projections of confocal sections and (D-F, H, K-N) show single confocal sections. (B) The overexpression of GFP-kkvΔWGTRE in a kkv mutant background does not rescue the absence of extracellular chitin deposition (white arrow, note the absence of CBP) and the protein accumulates in a generalised pattern. (C-D) The overexpression of GFP-KkvΔWGTRE does not produce intracellular chitin punctae, neither in trachea at early stages (C-C’) nor in salivary glands (D). (E-E”‘) GFP-kkvΔWGTRE colocalise with the ER marker KDEL. (F) GFP-KkvΔWGTRE does not colocalise with the marker FK2. (G) The overexpression of GFP-kkvΔCC in a kkv mutant background rescues the lack of extracellular chitin deposition in the trachea (note the presence of CBP staining). (H, I) The simultaneous expression of reb and GFP-kkvΔCC in salivary glands produces ectopic extracellular chitin (H), and no defects in trachea (I). (J) The overexpression of reb in trachea leads to morphogenetic defects. (K, L) Overexpressed GFP-Kkv localises mainly apically (orange arrowheads) although a bit of the protein can be detected in the basal region (yellow arrowheads). (M, N) Apical accumulation of overexpressed GFP-KkvΔCC is less conspicuous. (O) Quantifications of accumulation of GFP-Kkv and GFP-KkvΔCC in apical versus basal region. n corresponds to the number of ratios analysed (apical/basal ratio per cell), and brackets indicate the number of embryos used. Ratios were obtained from the apical (orange line in K) and basal (yellow line in K) domains of single cells in trachea and salivary glands. The underlying data for quantifications can be found in the S1 Data. Scale bars B, C, G, I, J: 25 μm; D-F, H, K-N: 10 μm., Peer reviewed

Polyploidy is an extended phenomenon in biology. However, its physiological significance and whether it defines specific cell behaviors is not well understood. Here we study its connection to macroautophagy/autophagy, using the larval respiratory system of Drosophila as a model. This system comprises cells with the same function yet with notably different ploidy status, namely diploid progenitors and their polyploid larval counterparts, the latter destined to die during metamorphosis. We identified an association between polyploidy and autophagy and found that higher endoreplication status correlates with elevated autophagy. Finally, we report that tissue histolysis in the trachea during Drosophila metamorphosis is mediated by autophagy, which triggers the apoptosis of polyploid cells., The work was supported by the Ministerio de Ciencia y Tecnologia from the Spanish Government and by the Generalitat de Catalunya., Peer reviewed

(A) Alignment of amino acids (aa) sequences of the isoform B of Exp (aa 356–433) and homologs; the blue square indicates 8 aa highly conserved, the red square includes 9 aa less conserved. (B-D) Show projections of confocal sections and (E-F) show single confocal sections. (B) The overexpression of expΔCM2 in an exp reb mutant background rescues the lack of extracellular chitin deposition. (C, D) The simultaneous expression of expΔCM2 and GFP-kkv produces morphogenetic defects in the trachea (arrowheads in C) and ectopic chitin deposition in the lumen of salivary glands (D). (E, F) Overexpressed Exp localises mainly in the apical region (orange arrowheads) with respect the basal domain (yellow arrowheads), while the apical accumulation of overexpressed ExpΔCM2 is less conspicuous (F). (G) Quantifications of accumulation of Exp and ExpΔCM2 in apical versus basal region. n corresponds to the number of ratios analysed (apical/basal ratio per cell), and brackets indicate the number of embryos used. Ratios were obtained from the apical (orange line in E) and basal (yellow line in E) domains of single cells in trachea and salivary glands. The underlying data for quantifications can be found in the S1 Data. Scale bars B, C: 25 μm; D-F: 10 μm., Peer reviewed

All images show projections of confocal sections, except D, I, L, O, and Q, which show single confocal sections. (A, B) The overexpression of GFP-kkv in the trachea leads to the presence of intracellular chitin vesicles at early stages (pink arrowheads) (A, A’). At later stages, intracellular chitin vesicles are not present, and chitin is deposited extracellularly in the lumen (blue arrowheads and inset) (B, B’). (C, C’) In exp reb mutants, the overexpression of GFP-kkv in the trachea produces intracellular chitin punctae until late stages (pink arrowheads). (D) The overexpression of GFP-kkv in salivary glands produces intracellular chitin vesicles (pink arrowheads). (E) Schematic representation of Exp protein. (F, G) In exp reb mutants, the expression of a wild-type form of exp/reb rescues the lack of extracellular chitin deposition (F, white arrow and inset), while expΔMH2/rebΔMH2 do not (G, white arrow points to absence of CBP). (H, I) The co-overexpression of GFP-kkv and expΔMH2 in control embryo does not produce morphogenetic defects in trachea (H) or extracellular chitin deposition in salivary glands (white arrow) (I); however, intracellular chitin punctae are present (pink arrowheads in H, I, and inset in H). (J) The coexpression of GFP-kkv and expΔMH2 in exp reb mutants produces intracellular chitin particles (pink arrowheads) but does not rescue the lack of extracellular chitin deposition. (K, L) RebΔMH2 localises apically in trachea (K) and in salivary glands (L). (M) MH2-reb is not able to rescue the absence of extracellular chitin deposition in exp reb mutants. (N, O) The simultaneous expression of MH2-reb and GFP-kkv does not produce morphogenetic defects or ectopic chitin deposition in trachea (N) and in salivary glands (white arrow in O), but intracellular chitin vesicles are present (pink arrowhead in O). (P, Q) MH2-Exp protein does not localize apically in trachea (P) or in salivary gland (Q). Scale bars A-C, F-H, J, M, N: 25 μm; D, I, K, L, O-Q: 10 μm., Peer reviewed

The excerpted portion of the immunoblot shown in the figures is highlighted by a black box., Peer reviewed

(A) Projection of confocal sections of salivary glands shows that Kkv is normally expressed in salivary glands and accumulates apically. (B-D) Single confocal sections of trachea at early stages. Single chitin punctae do not colocalise with deacetylases or Gasp. (E, F) Single confocal sections of salivary glands expressing serp and verm. Single chitin punctae do not colocalise with deacetylases. (G) Frames from live imaging movie 2 show that common GFP-Kkv and chitin punctae (yellow arrow) can separate from each other; many GFP-Kkv (green arrow) and chitin puncta (red arrow) do not colocalise. (H) The luminal GFP-Kkv punctae are labelled by GFP and Kkv. Scale bars A, E, F: 5 μm; C-D, G: 10 μm., Peer reviewed